ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs

Summary CRISPR-Cas9 technology coupled with human induced pluripotent stem cells allows precise disease modeling in pluripotent cells and subsequently derived specialized cell types. Here, we present an optimized CRISPR-Cas9 pipeline, ASSURED (affordable, successful, specific, user-friendly, rapid, efficient, and deliverable), to produce gene-modified single-cell-derived knockout or single-nucleotide-polymorphism-modified knockin hiPSCs clones. We describe steps for analyzing targeted genomic sequence and designing guide RNAs and homology repair template. We then detail the CRISPR-Cas9 delivery workflow, evaluation of editing efficiency, and automated cell isolation followed by clone screening.

5. Input gene name or ENSEMBL ID. 6. For example: SorCS1 or ENSG00000108018. 7. Make sure that the selected genome is correct. 8. For example: Homo sapiens GRCh38. 9. Set the destination folder to save the DNA sequence by creating new project folder. 10. Click on 'Import' to obtain the annotated gene sequence.
Note: Benchling will import the single transcript chosen for a gene which is the most conserved, highly expressed, has the long coding sequence and is represented in other key resources such as NCBI or Uniport. In case specific isoform information is required, or a knockout targeting all isoforms we recommend viewing gene information using GeneViewer on NCBI.
Note: the annotation of the full gene sequence will be a range of a chromosome sequence and should start with letters 'NG'. 16. Download GeneBank or FASTA file.
Note: choose the file format depending on your preferred sequence browser, i.e., SnapGene etc. 17. Using sequence viewer program (i.e., SnapGene or Benchling) identify the exons, introns, CDS, and protein translation.

hiPSC culture
Timing: 1 week Ensure that hiPSC cells used for gene editing are of excellent quality. Use the earliest available passage and make sure that the standard quality control has been completed. Each of our cell banks is subject to g-banding karyotyping and SNP analysis to test genetic stability, mycoplasma and sterility testing, morphology assessment and STR analysis to confirm cell line identity. 1 Additionally, we recommend testing for expression of markers for the undifferentiated state of hPSC (i.e., Oct3/4, Nanog, SSEA1, Tra-1-60) using immunofluorescence or FACS.
For our maintenance culture we routinely use Geltrex coating and Essential 8 media. However, certain cell lines might have specific media and matrix recommendations, i.e., mTeSR or StemFlex media, and vitronectin or Matrigel coating. During the process of gene editing, we switch from standard maintenance media to StemFlex or StemMACS (see key resources table), as we observe improved recovery after nucleofection and clone survival in these formulations.
Genetic engineering places the cells under selection pressure and requires several rounds of cell doublings until the edited clone is obtained. Therefore, to avoid accumulation of genomic instabilities, we recommend using cells 2-3 passages after thawing as close to the quality tested cell bank as possible. We also recommend maintaining hiPSCs and performing gene editing experiments in hypoxic conditions (37 C/5% CO 2 /5% O 2 ) as culturing hiPSCs under hypoxic conditions has several advantages, including enhanced pluripotency, increased proliferation, reduced oxidative stress, improved reprogramming efficiency, better differentiation potential and low frequency of genetic instabilities. 2,3 These benefits can result in improved hiPSC quality and functionality, which are crucial for downstream applications in regenerative medicine and disease modeling. General protocols describing plate coating, cell maintenance, and enzymatic and non-enzymatic dissociation have been described by Vallone et al. 4 Geltrex dilution and aliquots Timing: 1 h 18. Thaw Geltrex stock solution on wet ice at 4 C.
CRITICAL: The following steps must be performed on ice! All serological pipettes and tubes to be used should be pre-chill at À20 C for at least 20 min.
19. Transfer 45 mL of KO DMEM/F-12 to a 50 mL conical tube. 20. Add 5 mL of thawed Geltrex stock solution to the aliquoted KO DMEM/F-12. 21. Mix gently without introducing bubbles. 22. Quickly aliquot 1 mL into 1.5 mL Eppendorf tubes. 23. Place on ice immediately. 24. Transfer the aliquots into À20 C as soon as aliquoting is finished.
These aliquots must be further diluted immediately prior to application 1:12 in KO DMEM/F-12 to generate Geltrex Coating Solution.

gDNA isolation
Timing: 1 h Genomic DNA isolated from the parental cell line will be necessary for primer optimization, confirmation of genotype and identification of potential nucleotide polymorphisms in the sgRNA and HDR regions. Therefore, we recommend preparing this gDNA in advance from all the cell lines that will be subject to the genetic modification. It is possible to use gDNA isolated by crude methods (i.e., Phire Tissue direct kit or similar), but isolation on silica based columns or beads is preferable. We routinely use Qiagen and Promega DNA isolation kits.

MATERIALS AND EQUIPMENT
Alt-Râ S.p. HiFi Cas9 nuclease V3 aliquots Cas9 Nuclease from IDT is provided as 10 mg/mL solution. We recommend making 2 mL aliquots to avoid repeated thaw/freeze cycles.

StemFlex/CloneR2
During the cell cloning stage of the protocol, we recommend using StemFlex media supplemented with CloneR2 (StemFlex/CloneR2) to increase cell survival. CloneR2 promotes clonal survival and growth of hiPSCs during single-cell passaging. CloneR2 improves the efficiency of colony formation from single cells while maintaining the pluripotent state.
CloneR2 is diluted in media 1:10, for example, for 18 mL of StemFlex add 2 mL of CloneR2. Media supplemented with CloneR2 should be used up as soon as possible. Therefore, for economic reasons, we recommend preparing StemFlex/CloneR2 always fresh and in quantity specifically required by the application that is being performed on that day.
Similar results were obtained with StemMACS PSC-Brew XF human supplemented with StemMACS PSC-support XF human. Essential 8 media can also be used during gene editing protocol; however, we experienced reduced cell survival and clone recovery.

Design of guide RNAs
Timing: 30 min CRISPR can be used to generate gene knockouts (KO), single nucleotide (SN) modification, and insertions/replacement of larger DNA fragments (knockin, KI). 5 A major difference in design approach for these three applications is the selection criteria for gRNAs.
For SN editing and KIs, the gRNAs must be as close to the targeted site as possible. Therefore, the choice of potential gRNAs is frequently limited. On the other hand, gRNA selection for KOs is more flexible. In most cases, KOs can be generated through introduction of a single double-strand break (DSB) with a single gRNA or by using two gRNA near each other. Following DSB, non-homologous end joining (NHEJ) leads to random indel formation, with potential frame shift and, as a result, a premature stop codon occurrence or nonsense protein sequence. For a successful knockout, gRNA target sites must be in exons critical for protein function. Specifically, gRNAtarget sequences near the N-terminus should be excluded to avoid usage of an alternative start-site downstream of the annotated start codon. Similarly, gRNA-target sequences near C-terminus ought to be avoided, to maximize the chance of frame shifts and generation of a non-functional protein.
We suggest using Benchling and/or CRISPOR online tools for gRNA design for all applications. However, when a non-targeted KOs is required, we recommend the more straightforward Synthego tool.
The following steps describe design of gRNAs using online tools.
Note: As online tools change constantly and rapidly, we provide just an overview of the steps. Most online tools are self-explanatory and interactive. We encourage the users to explore the manuals and FAQ sections to find additional information. Below we describe design of gRNAs to create indels, and SNPs insertion in SORCS1 gene in Benchling. For example: exon 4 of SORCS1 for KO ( Figure 1A) or exon 25 for SNP ( Figure 1B).
Note: make sure to select $30 bases before and after coding sequence to capture all the gRNA available in the region 6. Click on '+' to create the guide sequences. 7. Benchling will generate a list of gRNAs available in the selected region with On-Target and Off-Target score values ( Figures 1A and 1B).
Optional step: If working with CRISPOR, follow the below steps.
8. Input the sequence in the designated window. 9. Select genomic assembly: GRCh38.
Note: Genome assemblies are updated continuously, thus, make sure to use the latest version.
Alternative: If gRNAs are for KOs and the specific target region does not need to be considered, we recommend using the Synthego CRISPR Design Tool. This tool maximizes knockout probability by targeting an early part of the gene in an exon common to all transcript variants. All gRNAs are ranked according to their descending on-target activity and ascending offtarget potential. In principle, one gRNA is sufficient to efficiently introduce an indel. However, in silico tools are still limited in predicting the cutting efficiency of gRNAs. 6 Therefore, we recommend choosing 3-4 top scoring gRNAs and testing them in vitro.

Selection of gRNAs
Timing: 30 min We recommend selecting multiple gRNAs in the proximity of the desired modification. For KOs, up to 4 gRNAs can be used at the same time to increase the chance of indel formation. For KIs, use of 2 gRNAs simultaneously can increase probability of HDR. 7 However, increasing number of gRNAs also increases potential off-target effects and in case of KIs can require introduction of additional silent mutations in the repair template. Therefore, the number of gRNAs used per modification should be carefully considered and will depend on the specific gene and targeted region. In the section delivery of CRISPR components to iPSCs, we describe delivery of 1 or 2 gRNAs. Note: this will increase the probability of A or T duplication. 9 b. G at 1st position upstream of the PAM sequence; Note: gRNAs with G at 1st position have higher cutting efficiencies as compared to gRNAs with T at this position. 10 Note: It is not always possible to fulfil all the above criteria for gRNAs.
CRITICAL: If the SNP is located outside of the seed region or corresponds to the 'N' of the 'NGG' PAM site, then the modified allele after HDR will be subject to re-cleavage by Cas9 RNP. Therefore, silent mutations should be introduced to either seed sequence or PAM sequence in the single-stranded oligodeoxynucleotide (ssODN) template to prevent recutting. When these additional mutations are not introduced, the number of screened clones needed to obtain the wanted modification can increase substantially. For details see section: design of ssODN for KIs.

Design of primers
Timing: 30 min As each hiPSC line is derived from an individual donor, there is a high probability of SN variations (SNV) occurrence in any given DNA region ( Figure S2). Therefore, we recommend performing Sanger sequencing of the PCR-amplified targeted region before proceeding with gRNAs and ssODNs ordering. The primers used for this initial query can also be used for the future verification of the gene editing success and for clone screening. 18. Select approximately 250bp up-and downstream sequences from the target site.
Note: Large deletions that expand past the fragment covered by the primers are possible as described by Simkin et al. 11 Therefore, it might be required to use a primer pair spanning a larger region around target site. However, generation of hemizygotes rather than homozygotes is less likely to occur when RNPs are used, rather than plasmids.
19. Use Primer Blast design tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to design primers that will amplify 400-450bp of DNA with cut site position as close to the middle. 20. Select and order two primer pairs (Table 1, Figures S1A-S1B).
Note: To accurately estimate gene editing efficiencies and further select edited clones the quality of the PCR product and Sanger sequencing must be high. Therefore, we recommend choosing at least two primer pairs.

Design of ssODN for KIs
Timing: 30 min CRITICAL: It is critical to prevent Cas9 nuclease from recutting the modification site after the HDR. The strategy will depend on the choice of gRNA and the SNP. [15][16][17] For example, if the SNP to be introduced already lies in the ''GG'' of the PAM, then additional mutations might not be necessary, since the recutting post HDR will be limited. If not, a silent mutation of codons covering the PAM sequence (one of the GG) should be introduced if possible; alternatively, the last five nucleotides of seed sequence can be mutated to prevent gRNA from binding. [18][19][20][21][22] Below, we describe the design of ssODN using Benchling.
25. In the gene viewer in Benchling, select CRISPR icon on the right side (crosshair icon). 26. Select: Open HR template design and follow prompts. 27. The program will create the copy of the sequence. 28. Following the prompts in the right-side panel, insert the SNP sequence to be knocked in. 29. Adjust the HA as described in step 22. 30. Click Next. 31. The program will suggest mutations to the DNA template to prevent sgRNA from binding and recutting.
Note: It is not always possible or desirable to introduce additional mutations. When designing HDR template without help of professional software (i.e., Benchling), we recommend to consult codon usage tables when introducing silent mutations. For example: https://www. genscript.com/tools/codon-frequency-table 32. Select the non-PAM strand as donor template as described in step 23.
Note: Here we described template design for introduction of 1-5 base pair changes. However, it is also possible to introduce small tags using ssODN as templates. We have successfully integrated double HA ($60bp) and triple Flag tag ($70bp) at endogenous loci using ssODN templates. In these cases, the total template length was extended to 140 and 170 bp respectively.

PCR optimization
Timing: 8 h ll

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Due to unpredictability of PCR efficiency at different genomic loci we recommend testing both primer pairs designed in Steps 18-20 with at least three different polymerases i.e., PHIRE, AmpliTaq, and/or Kappa2G, and/or GXI polymerase, and/or Q5) at three annealing temperatures. Use gDNA from before you begin section: gDNA isolation.
33. Prepare 65 mL (3 3 20 mL rxns + 5 mL dead volume) of master mix for each polymerase and primer pair according to the polymerase specifications (Tables 2-4), total 6 master mixes: 2 primer pairs 3 3 polymerases. 34. Aliquot 20 mL of each polymerase/primer pair master mix into 3 PCR tubes, total 3 3 6 master mixes: 18 tubes. 35. Use one tube from each polymerase/primer master mix for 3 different annealing temperatures (Table 5). Set up the thermocycler for respective conditions as in example (Table 6) 36. Analyze PCR reactions by agarose (1%-2%) gel electrophoresis. 37. Choose the condition with the clearest band and no off-target bands. For example, for each primer pair in SorCS1 exons 4 and 25 the clearest bands were obtained with PHIRE polymerase with 62 C annealing temperature (Figures S1C-S1D). 38. Repeat the PCR with the chosen condition. 39. PCR purify or gel extract the PCR product.
Note: Choose any of the standard PCR or gel extraction methods that provide sufficient purity for Sanger sequencing.
40. Perform Sanger sequencing on the PCR product with forward primer, the same one used for PCR. 41. Analyze the sequencing by comparing the obtained sequence with the chosen genome reference.
Note: As hiPSC lines are derived from various individual donors, it is not unlikely to find single nucleotide polymorphisms which could interfere with gRNA binding sites ( Figure S2).

Synthesis of CRISPR components
Timing: 1-2 weeks  Once the target sequence is analyzed and no SNP interfering with the gRNA and HDR template are detected, CRISPR components can be purchased. They are readily available from many vendors, we routinely obtain HiFi Cas9, gRNAs, and ssODNs from IDT (https://eu.idtdna.com/pages/products).
CRITICAL: The following steps describe the procedure using the components from IDT, when using a different source of gRNAs and ssODN templates follow the manufacturer instructions for reconstitution and storage. 46. Store all CRISPR components at À20 C. The day before delivery of CRISPR components (Day -1), the cells from the section before you begin: hiPSC culture must be re-plated ( Figure 3). This step helps to synchronize the cell cycle and promote most of the cell to be in S-phase, when HDR pathways are most active. 23 The number of wells needed to begin the experiment is dependent on the number of nucleofections performed. Enough cells to plate 2 wells of a 6WP with 2 3 10 5 cells each (one for one nucleofection and one for untransfected control) is needed (Figure 3). Usually one $50% confluent well of hiPSCs is enough to collect $1 3 10 6 cells. However, we recommend evaluating cell number per well before performing the experiment.

Delivery of CRISPR components to hiPSCs
Timing: 3-4 h The following procedure describes the cell preparation and transfection procedure for gRNA, Cas9 protein, +/-ssODN (see Table 7 for options) using Neon nucleofector and Neonä Transfection System 10 mL Kit. Alternative electroporation/nucleofection protocols can also be applied with appropriate optimization. For any additional gRNAs that are transfected separately the following procedure must be scaled accordingly. Note: for each transfection prepare 2 receiving wells of a 6WP, and one well for untransfected cells; in this example 3 wells (Figure 3).   Optional step: For SNP KI incubate cells at 32 C/5% CO 2 /5% O 2 for 48 h to improve HDR efficiency up to 2 fold. 24 Optional step: For SNP KI use 1 mM AltR HDR enhancerV2 for 48h after transfection. For example, supplement 2 mL of Stem Flex/CloneR2 media with 2.8 mL of IDT HDR enhance v2 (1 mM) per 1 well of 6 well plate.
Note: we recommend to also use HDR enhancer in combination with 'cold-shock'. We observed $ more than 7-fold increase in KI-score value when using HDR enhancer (n = 1).

After 48 h, change media to StemFlex media without Clone R2.
Note: after removal of CloneR2 cell morphology will change; while CloneR2 is present in the media cells appear slightly more spread out and have less rounded morphology (more spindle-like). However, the overall morphology of hiPSCs remains like that of typical human ll OPEN ACCESS STAR Protocols 4, 102406, September 15, 2023 pluripotent stem cells, characterized by colonies with well-defined borders and individual cells with a high nucleus-to-cytoplasm ratio.

Timing: 24 h
Gene editing efficiency can be assessed in the bulk sample based on the distribution of the sequencing chromatogram from *.ab1 files. Freely available online deconvolution software compares sequencing obtained from unedited cells with sequencing from mixed edited cell population and discerns the percentages of different indels and/or HDR incorporation. The most commonly used tools include: Synthego ICE Tool (https://ice.synthego.com/#/) and TIDE (https://tide.nki.nl/). Such tools are not exact but offer good approximation of the editing efficiency, which in turn can help to decide on the number of clones to be screened. For example, if predicted editing efficiency is 50%, screening of 30 clones is sufficient to yield 15 clones with desired modification. On the other hand, if editing efficiency is only 5% it would be necessary to screen 300 clones to arrive at the same number of edited clones.
Here we describe analysis with Synthego tool. Note: make sure that the colonies begin to detach but not completely.
e. Aspirate EDTA slowly. f. Add 2 mL of BambankerTM freezing medium to the well. g. Using a 5 mL pipette dislodge the cells gently by pipetting.
Note: Do not pipette up and down more than 5 times not to break up the clumps.
h. Transfer the cell suspension into 2 cryovials. i. Place the cryovials immediately into the freezing container and at À80 C overnight. j. Transfer the cells to a liquid N2 tank the next day.

Optional
Step: If the editing efficiency is evaluated (Steps 82-88) before the cells reach 75% confluency, the cells can be plated directly for single cell clone isolation (see section single cell clone isolation) without freezing.
Step 81 can be omitted. Alternatively, cells remaining after clonal plating can be frozen.
82. Use gDNA A and C from step 79 to perform PCR using conditions chosen in step 37.
Note: use 30 mL reaction volume.
Optional step: Resolve 10 mL of each of the PCRs (A and C) by 1%-2% agarose electrophoresis to make sure the PCR generated bands of expected size. a. alignment of the control and edited sample traces (Figures S3A and S3C). b. success rates for KO, KO score, defined as proportion of indels that indicate a frame shift or are 21+bp in length ( Figure S3B) or KI efficiency, a KI score, defined as proportion of sequences that indicate the knockin insert ( Figure S3D). c. Indel and/or HDR distribution.

Optional
Step: When performing larger excisions (i.e., whole exon), it might be sufficient to assess the efficiency of editing by resolving PCR products on the gel.

Timing: 2 weeks
Once the editing efficiency is assessed, cells can be plated for single cell clone isolation. Here we describe the single cell cloning using isoCell single cell cloning platform. However, other methods can be applied, i.e., limiting dilutions and clone picking. The following procedure is described for plating one cloning plate (256 individual wells), with expected outgrowth efficiency of $40%, resulting in $100 individual clones. The procedure is as described 4 with modifications.

Optional
Step: If the cells were not frozen but remain in the well B (Step 80), omit Step 81 and Step 89. Proceed directly with Step 90. 89. 48 h before planned single cell seeding (Day -2), thaw cells frozen in step 81: a. Add 1 mL of Geltrex Coating Solution (see materials and equipment section) to 1 well of a 6WP. b. Incubate for 1 h at 37 C/5% CO 2 incubator. c. Remove cryovial from LN2 and place on dry-ice. d. Prepare 10 mL of StemFlex supplemented with 650 mL of CloneR2 (1:20). e. Aliquot 8 mL of StemFlex/CloneR2 to a conical tube. f. Quickly thaw frozen cells by submerging the vial in 37 C water bath. g. Wait for almost all the ice to melt. h. Transfer the cell suspension slowly to the conical tube using P1000.  ii. In a 1.5 mL Eppendorf tube, prepare 500 mL of cell suspension in StemFlex/CloneR2 at a concentration of 7,500-10,000 cells/mL. iii. Add 4.4 mL of iMatrix-511 (0.025 mg/cm 2 ). iv. Place the tube with cells on the isoCell. b. To plate single cells into GRID chambers run the 'Plate' program ('Isolate' menu tab). c. Follow prompts on the isoCell display. 92. After the cells are dispensed into the GRID, incubate the plate at 37 C for 20-30 min. 93. Using isoHub, identify and select the chambers with single cells.

Optional
Step: If isoHub is not available, perform the identification of chambers with single cell manually and input the data into isoCell using the 'Input' program ('Isolate' menu tab).
CRITICAL: do not let the chambers reach more than 1 / 2 of the surface area, if cells overgrow the media volume is not sufficient to support cell survival 100. Prepare two 96-well plates for each 96 clones to be harvested.

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Note: Each clone will be split into 2 wells of a 96WP to create mirror plates. Therefore, for each clone ready to be harvested coat one well in each 96WP.
a. Add 50 mL of Coating Solution (see materials and equipment section) to required number of 96 wells. For example, for 36 clones, coat 72 wells: 36 wells in each of the 96WPs. b. Incubate at 37 C, 5% CO 2 for 1 h. c. Prepare StemFlex/CloneR2. Note: Required volume will be dependent on the number of clones, but generally each clone requires 320 mL of media. For example, for transfer of 32 clones, 12 mL of media is required. 107. Perform PCR on the gDNA extracted from clones as optimized in step 37. 108. Perform Sanger sequencing on PCR products using forward primer. 109. Perform KO or KI analysis as described step 85-88 ( Figures S4-S5).
CRITICAL: Carefully analyse sequencing and Synthego ICE results for analysis of the clones. Some clones will remain WT ( Figures S4A and S5A), some clones will be heterozygous with different repair for each allele ( Figures S4B and S5B), and some clones will be homozygous for the intended modification ( Figures S4C and S5B). For more details see expected outcomes section.

Timing: 1 h
Clone freezing in 96WP allows for a brief pause for the PCR screening to be completed. It is not recommended to store clones in this format for extended period of time, as 96WP cannot be transferred to LN2. Note: as transfer to liquid N2 is not possible, the cells in the plate can only be stored short term (up to 6 weeks).

Preparation of seed banks
Timing: 1 week As genetic manipulation, clonal selection and expansion generates selection pressure and can accelerate emergence of unwanted genetic instability, we strongly recommend performing a small screen to select only genetically stable clones prior to further pluripotency characterization. The following protocol describes thawing, expansion, and Generation of seedbanks from 5-6 positive clones. This procedure allows for faster turn over in case some of the clones have undesirable karyotype profiles. CRITICAL: Following gene editing pipeline the resulting clones should be subjected to standard iPSC quality control to ensure that no genetic abnormalities occurred during the editing and cloning process. We recommend expanding the selected clone, freezing a bank ($50 vials) and performing SNP array analysis and karyotyping to assess genetic stability. Whenever possible, we recommend banking at least two edited clones. Additionally, a routine assessment of morphology and expression of markers of undifferentiated cells should be conducted. Confirmation of genetic modification by PCR and Sanger sequencing or using NGS should also be performed on final bank. We also highly recommend assessing the integrity of the 5-10 top off-target sites for each of the gRNAs used.

EXPECTED OUTCOMES
Design and selection guidelines for gRNA and HDR template described above are universal and should allow for successful targeting of most of the genomic loci. Primer optimization (Steps 18-22, 33-42) prior to editing experiment allows us to reduce time and effort during clone screening and ensures good quality of sequencing for clone validation. Introduction of freezing pause steps (Step 81) allows for more flexibility.
We have successfully applied the ASSURED protocol to target 32 gene loci across 22 different iPSC lines ( Figure 4B). On average, we obtained KO-score of 79.5 G 23. KI efficiency is generally lower than for KO, with an average KI-score of 57.5 G 27 ( Figure 4B). The optimized cloning method using IsoCell resulted in $70 single cell containing chambers per one 256-chamber plate as scored on the day of plating (Step 92, Figure 4A) and out of those on average 46 (65%) clones survive ( Figure 4A). The lowest KO and KI scores we encounter were 15 and 10 respectively ( Figure 4B). Despite that, due to relatively high cloning efficiency, up to 5 clones were obtained from just one 256-chamber plate.

LIMITATIONS
Although we have not yet encountered such a case, for certain genetic loci in combination with specific hiPSC lines it might be impossible to identify a suitable gRNA or perform successful HDR. In such cases, excisions of larger fragments (i.e., whole exon) and subsequent repair with larger templates might be an option. Some cell lines might be more sensitive to single cell cloning, requiring optimization of cloning strategy. In cell lines with higher susceptibility to spontaneous differentiation, it might be necessary to generate seed banks from more clones, to identify the most stable clones.
Finally, targeting genes controlling proliferation or cell survival, especially for KO, might result in growth disadvantage or cell death of the successfully edited clones. Therefore, targeting certain genes might not be biologically feasible.

Potential solution
Identify gRNAs targeting upstream and downstream of the target site. Design longer HDR and perform large KI. It is advisable to perform gRNA cutting efficiency testing prior to including HDR in the transfection.
Poor cell survival after nucleofection (steps 61-80) Cell lines do not attach after nucleofection or die in subsequent days.

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Potential solution Some iPSC lines are sensitive to culture condition changes or exposure to certain chemicals (AltR HDR enhancer, etc.) and therefore pre-adapt the cell line to the Stem flex media conditions and optimize HDR enhancer concentration. Additionally, optimization of cell numbers used or the voltage for nucleofecting might be necessary.
Low gRNA cutting efficiency (steps 85-88) After bulk analysis there are few indels and/or low KO score.

Potential solution
On-target scores for gRNAs provided by CRISPR design software are derived purely in silico. Therefore, it is possible that in vitro the gRNAs cutting efficiency is not as high as predicted. We recommend selecting additional gRNAs in the region, if available, or adapting the strategy as described for 'No gRNAs directly at the editing site'.

Potential solution
If not done previously, add HDR enhancers. Additionally, redesign the HDR template by changing the asymmetry ratio, target strand or length of homology arms. Use HDR template mixes; mix HDR templates with different symmetries, or homologous to either of the strands.
Low cloning efficiency (steps 91-99) Very few clones survive the single cell cloning step.

Potential solution
Optimize the culture conditions for the specific iPSC line. Some iPSC lines are more sensitive to single cell growth conditions. It might be necessary to test other media and/or coating of the grid chambers.
No clones positive for the desired modification (steps 107-109) Editing efficiency was high based on bulk sequencing, but no positive clones were identified after clonal seeding.

Potential solution
Designed edit results in growth disadvantage or cell death. Reevaluate biological function of the target gene and check if there is no indication that gene modification can be lethal or detrimental to cell proliferation. In specific case of iPSCs alterations to pluripotency genes may lead to undesirable differentiation.

Too many KI homozygous clones (steps 107-109)
The project requires heterozygous and homozygous clones, but all clones identified with the HDR incorporation are homozygous.

Potential solution
Repeat the experiment including WT HDR template. Use 1:1 mixture of WT and mutant templates to increase the chance of obtaining heterozygous clones.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Sebastian Diecke (Sebastian.Diecke@mdc-berlin.de) and technical contacts, Katarzyna Anna Ludwik (Katarzyna.ludwik@bih-charite.de) and Narashima Telugu (NarasimhaSwamy. Telugu@mdc-berlin.de).

Materials availability
No unique reagent was generated in this study.

Data and code availability
No unique datasets or data were generated in this study.